酵母菌生產穀胱甘肽之最適化條件研究

穀胱甘肽 (Glutathione，GSH) 為一種天然抗氧化劑，並具有維持細胞內氧化還原之平衡、提升免疫力及解毒等多種功效，其可於酵母菌發酵過程中於細胞內產生。在現今，提高酵母菌細胞中GSH含量為現今工業研究重點之一，GSH可應用於食品工業 (如：增味劑)、醫學治療 (如：抗衰老、抗細胞凋亡) 及化妝品工業 (如：皮膚美白)等。本研究第一部份自實驗室挑選10株酵母菌菌株，其中5株為Saccharomyces cerevisiae，另外5株則為Non-S. cerevisiae，將其分別進行5種萃取方法 (酸水解萃取法、乙醇萃取法、水浴萃取法、冷凍解凍萃取法及細胞破碎萃取法)，探討其GSH含量及固定細胞乾重下之GSH含量百分比 (以下簡稱GSH萃取率)，篩選出較佳之萃取方法及菌株。第二部分為將所篩選出之菌株，進行基本生長特性探討及耐受性試驗。第三部分則為，探討菌株生產GSH含量之最適化條件，以一次一因子法探討不同添加濃度之葡萄糖、半胱胺酸及檸檬酸鈉其因子階層，並利用反應曲面法 (Response surface methodology, RSM) 之Box-Behnken設計 (BBD)，探討酵母菌生產GSH之最適條件。結果顯示，10株酵母菌分別經由5種萃取方法 (酸水解萃取法、乙醇萃取法、水浴萃取法、冷凍解凍萃取法及細胞破碎萃取法) 萃取後，以S. cerevisiae G35及P13於乙醇萃取法中之GSH含量及GSH萃取率為最高，其中S. cerevisiae G35之GSH含量為15.34 mg/L，GSH萃取率為1.32 ± 0.02%；而S. cerevisiae P13之GSH含量則為13.22 mg/L及GSH萃取率為1.02 ± 0.01%。接著探討S. cerevisiae G35及P13於耐受性試驗中之表現，不論是在耐酸性或耐膽鹽試驗中，其GSH含量皆偏低，僅在pH 4.5之第24小時，S. cerevisiae G35及P13中有較佳之GSH含量 (18.28 mg/L及18.80 mg/L)，最後於RSM-BBD探討其最適化條件，S. cerevisiae G35於葡萄糖添加濃度16.17%、半胱胺酸添加濃度10.6 mmol/L及檸檬酸鈉添加濃度7.05 g/L，可得最佳GSH含量89.91 ± 0.66 mg/L；而S. cerevisiae P13則為葡萄糖添加濃度16.17%、半胱胺酸添加濃度10.6 mmol/L及檸檬酸鈉添加濃度7.05 g/L，可得最佳GSH含量78.81 ± 1.03 mg/L，S. cerevisiae G35及P13之GSH含量與尚未優化之GSH含量分別提升486.11%及496.14%。綜上所述，葡萄糖、半胱胺酸及檸檬酸鈉之添加確實能提升酵母菌中GSH含量，可將此三種實驗因子應用於日後酵母菌中GSH之生產，然而，若要應用於機能性產品中可能需要在其產品外覆上一層腸溶衣，保護其不受胃腸道中酵素破壞，以發揮GSH之效能。

關鍵字

Saccharomyces cerevisiae ；穀胱甘肽；反應曲面法；抗氧化

並列摘要

Glutathione (GSH) is a natural antioxidant that has multiple functions such as maintaining intracellular oxidation-reduction balance, improving immunity, and detoxifying. It can be produced in cells during the fermentation process of yeast. Nowadays, increasing the GSH content in yeast cells is one of the focuses of current industrial research. GSH can be used in food industry (such as: flavor enhancer), medical treatments (such as anti-aging, anti-apoptosis) and cosmetics industry (such as skin whitening). In the first part of this study, 10 of yeast strains were selected from the laboratory, 5 of which were Saccharomyces cerevisiae and the other 5 were Non-S. cerevisiae, then respectively conduct to 5 extraction methods (acid hydrolysis extraction, ethanol extraction, water bath extraction, freezing/defrosting extraction and cell disruption extraction), explore its GSH content and GSH extraction rate, and then screen out better extraction methods and strains. The second part was discuss the basic growth characteristics and tolerance test of strains. The third part is to explore the optimal conditions for strain production of GSH content, explore the factor hierarchy of different concentrations of glucose, cysteine and sodium citrate, and using response surface methodology (RSM) of Box-Behnken design (BBD) to explore the optimal conditions for yeast production of GSH. The results showed that 10 yeast strains were extracted through 5 extraction methods (acid hydrolysis extraction, ethanol extraction, water bath extraction, freezing/defrosting extraction and cell disruption extraction), and then S. cerevisiae G35 and P13 their GSH content and extraction rate in ethanol extraction were the highest. The GSH content and extraction rate of S. cerevisiae G35 were 15.34 mg/L and 1.32 ± 0.02%; while the GSH content and extraction rate of S. cerevisiae P13 were 13.22 mg/L and 1.02 ± 0.01%. Next, the performance of S. cerevisiae G35 and P13 in the tolerance test was discussed. Whether in the acid resistance or bile salt resistance test, the GSH content was low. Only at the 24th hour of pH 4.5, S. cerevisiae G35 and P13 has better GSH content (18.28 mg/L and 18.80 mg/L), Finally, the optimal conditions were discussed in RSM-BBD. The optimal GSH content of S. cerevisiae G35 was 89.91 ± 0.66 mg/L when the glucose concentration was 16.17%, the cysteine concentration was 10.6 mmol/L, and the trisodium citrate concentration was 7.05 g/L; while S. cerevisiae P13 added glucose concentration of 16.17%, cysteine concentration of 10.6 mmol/L and trisodium citrate concentration of 7.05 g/L, can obtained optimal GSH content of 78.81 ± 1.03 mg /L, the GSH content of S. cerevisiae G35 and P13 compare with the GSH content that has not been optimized respectively increased by 486.11% and 496.14%. In summary, the addition of glucose, cysteine and trisodium citrate can increase the GSH content in yeast. These three experimental factors can be applied to the production of GSH in yeast in the future. However, if it is to be applied to functional products may need to be coated with an enteric coating to protect them from damage by enzymes in the gastrointestinal tract in order to maximize the effectiveness of GSH.